Efficient vaccines must not only stimulate innate immune receptors, but also deliver antigens to specific subcellular compartments so that they can be processed via the class I and class II antigen-presenting pathways and lead to the production of antigen-specific cytotoxic T cells and antibodies (Amanna and Slifka, 2011, Virology 411:206-215). Live replicating pathogenic bacteria, such as Salmonella typhimurium, rendered avirulent and engineered with the ability to express foreign antigens are being considered as vaccine vectors to protect against various infectious diseases or as therapeutic agents against cancer (Curtiss et al., 2010, Crit Rev Immunol. 30:255-270; Hegazy and Hensel, 2012, Future Microbiol. 7:111-127; Moreno, et al., 2010, Curr Gene Ther. 10(1):56-76). Although they potently stimulate innate immune receptors, one limitation of these bacterial vaccine systems is their inefficient capacity to stimulate cytotoxic T cell responses (Gao et al., 1992, Infect. Immun. 60:3780-3789; Yang et al., 1990, J. Immunol. 145:2281-2285), which require the delivery of antigens to the cytosol of antigen presenting cells. This limitation has been largely overcome by the use of type III secretion systems (T3SS) (Chen et al., 2006, Infect Immun 74:5826-5833; Russmann et al., 1998, Science 281:565-568), which are complex multi-protein molecular machines that deliver bacterial virulence effector proteins into the host cell cytosol (Galan and Wolf-Watz, 2006, Nature 444:567-573). Proteins destined to travel the T3SS pathway possess discrete signals that direct them to the secretion machine (Arnold et al., 2010, Microbes Infect. 12:346-358). When incorporated into heterologous proteins, these signals can target virtually any protein for delivery through this secretion pathway (Chen et al., 2006, Infect Immun 74:5826-5833; Michiels and Cornelis, 1991, J. Bacteriol. 173:1677-1685). Consequently, T3SS have been engineered to deliver heterologous antigens in the context of virulence attenuated bacterial pathogens (Russmann et al., 1998, Science 281:565-568). Heterologous protein antigens delivered by this system have been shown to stimulate antigen specific CD8+ T-cells, which in animal models conferred protection to a variety of infectious diseases or caused the regression of established tumors (Russmann et al., 1998, Science 281:565-568; Russmann et al., 2001, J Immunol 167:357-365; Wieser et al., 2012, Int J Med Microbiol. 302:10-18; Zhu et al., 2010, Cancer Science 101:2621-2628; Chamekh, 2010, Immunopharmacol Immunotoxicol. 32:1-4; Tartz et al., 2008, Vaccine 26:5935-5943; Nishikawa et al., 2006, J. Clin. Inv. 116:1946-1954; Konjufca et al., 2006, Infection and Immunity 74:6785-6796; Evans et al., 2003, J. Virol. 77:2400-2409; Kotton et al., 2006, Vaccine 24:6216-6224; Sevil et al., 2008, Vaccine 26:1879-1886). A second limitation of the virulence-attenuated bacterial vaccine systems relates to their safety. Residual virulence of the attenuated pathogen or the potential for virulence-reversion may limit their use in certain populations such as children or the immunocompromised.
Since protein translocation by T3SSs requires energy provided by ATP hydrolysis and a proton gradient (Galán, 2008, Nat Struct Mol Biol. 15:127-128), the use of this system has been restricted to the context of live virulence-attenuated bacteria. The intrinsic complexity of the assembly of this large, multi-protein machine combined with its energy requirements, preclude the development of a synthetic system capable of supporting the function of T3SS.
Therefore, there is a need in the art for compositions and methods for an efficient non-replicating antigen-delivery system that can be used for vaccines and immunotherapy. The present invention satisfies this unmet need.